Type-I four-helix bundle cytokines mediate a wide range of physiological processes through their effects on cell growth, differentiation, and proliferation. Collectively, through both pleiotropic and unique activities, they contribute to the homeostasis of the immune, hematopoietic, and nervous systems. The majority of hematopoietic and immune regulatory cytokines act through a small group of shared transmembrane signaling receptors. The two most widely used shared receptors are gp130, and the common gamma chain (3c), which engage their cytokine ligands through their extracellular regions, resulting in activation of intracellular JAK/STAT signaling cascades. Gp130 is the shared receptor for eleven cytokines (IL-6, IL-11, Ciliary Neurotrophic Factor, Oncostatin, Leukemia Inhibitory Factor, and others), and 3c is the shared receptor for 6 cytokines (IL-2, -4, -7,- 9, -15, -21). During the prior term of this award, we elucidated structural architectures of the two paradigmatic receptor complexes in each family, gp130/IL-6 and 3c/IL-2. These ternary and quaternary complexes represent blueprints for the assembly of receptor-ligand complexes used by all ligands in each shared receptor family. In this proposal, we escalate our investigations into the structural biology of these shared receptors in regards to extracellular ligand recognition, assembly of hetero-oligomeric cell surface complexes, transmembrane signaling, and intracellular activation of JAK and STAT. In order to do this, we are: 1- determining x-ray crystal structures of soluble extracellular multimeric receptor-ligand complexes, 2- measuring the cooperativity and assembly energetics of stepwise cytokine-receptor complex formation, 3- purifying the entire full-length receptor complexes, as membrane proteins, bound to both cytokine and intracellular adaptors and imaging them by electron microscopy, 4- reconstituting the intracellular signaling complexes consisting of JAK and STAT, and 5- complementing the crystallographic studies with NMR to interrogate potential allostery during signaling. In this fashion, by taking a multi-disciplinary strategy we propose to obtain a complete molecular snapshot of a shared signaling receptor from the initial engagement of ligand through the activation of intracellular signaling cascades. PROJECT NARRATIVE: Growth factors termed cytokines, and their receptors, are essential to the normal functioning of the immune, hematopoietic, and nervous systems, and dysfunction of the growth factor network underlies numerous human disease conditions such as autoimmunity and cancer. We propose to use the techniques of biochemistry and structural biology to visualize the three-dimensional shapes of growth factors bound to their cellular receptors, and understand how this binding event is communicated across the cell membrane. These studies will give us insight into basic receptor signaling mechanisms, as well as facilitate the manipulation of the cytokine receptor system for therapeutic purposes.